The overall aim of the project is to define the reactions between antithrombin III with thrombin and other procoagulants, and to elucidate the mechanism by which heparin inhibits blood coagulation. Since antithrombin and heparin affect a number of reactions, results of these studies should contribute greatly to the understanding of the regulation of blood coagulation. Having isolated biologically active heparin fractions with defined molecular weight ranges and studied their effect on the thrombin-antithrombin reaction, during the next year we will investigate the relationship between molecular weight, thrombin-inhibiting activity, and activity in the antithrombin-factor Xa reaction. Thus, the aim will be to determine whether the same structural features are required for inhibiting thrombin and factor Xa. Similar studies will be conducted with heparitin sulfate. The binding properties of heparin and structural location of the active site will be investigated by means of fluorescent-labeled heparins and immobilized heparins that we prepared during the last year. It was shown in previous years, that heparin inhibits the neutralization of thrombin by alpha 1-protease inhibitor and that this effect is the result of the binding of heparin with thrombin. This action of heparin is related to its molecular weight. During the coming year, therefore, it is planned to conduct quantitative studies on the relationship between the molecular weight of heparin and its binding to thrombin. These investigations should also clarify some aspects of the action of heparin in the thrombin-antithrombin reaction.